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Abstract

Background

The development of analgesic tolerance following chronic morphine administration can
be a significant clinical problem. Preclinical studies demonstrate that chronic morphine
administration induces spinal gliosis and that inhibition of gliosis prevents the
development of analgesic tolerance to opioids. Many studies have also demonstrated
that ultra-low doses of naltrexone inhibit the development of spinal morphine antinociceptive
tolerance and clinical studies demonstrate that it has opioid sparing effects. In
this study we demonstrate that ultra-low dose naltrexone attenuates glial activation,
which may contribute to its effects on attenuating tolerance.

Results

Spinal cord sections from rats administered chronic morphine showed significantly
increased immuno-labelling of astrocytes and microglia compared to saline controls,
consistent with activation. 3-D images of astrocytes from animals administered chronic
morphine had significantly larger volumes compared to saline controls. Co-injection
of ultra-low dose naltrexone attenuated this increase in volume, but the mean volume
differed from saline-treated and naltrexone-treated controls. Astrocyte and microglial
immuno-labelling was attenuated in rats co-administered ultra-low dose naltrexone
compared to morphine-treated rats and did not differ from controls. Glial activation,
as characterized by immunohistochemical labelling and cell size, was positively correlated
with the extent of tolerance developed. Morphine-induced glial activation was not
due to cell proliferation as there was no difference observed in the total number
of glial cells following chronic morphine treatment compared to controls. Furthermore,
using 5-bromo-2-deoxyuridine, no increase in spinal cord cell proliferation was observed
following chronic morphine administration.

Conclusion

Taken together, we demonstrate a positive correlation between the prevention of analgesic
tolerance and the inhibition of spinal gliosis by treatment with ultra-low dose naltrexone.
This research provides further validation for using ultra-low dose opioid receptor
antagonists in the treatment of various pain syndromes.

Background

Opioid drugs, such as morphine, are widely used for the management of moderate to
severe pain. Unfortunately, the usefulness of morphine and other opioid analgesics
in the management of pain is limited due to the development of tolerance to the analgesic
effects of these drugs with repeated exposure [1]. Clinically, the onset of tolerance necessitates increasing doses of opioids, which
in turn typically increases the number and severity of adverse effects and compliance
[2].

Morphine acts to inhibit nociception predominately via Gi protein-coupled μ-opioid receptors [3,4] located in nociceptive pathways throughout the central nervous system including the
dorsal spinal cord. Within the spinal cord, μ-opioid receptors are well recognized
to localize on pre- and post-synaptic nociceptive neurons, but they are also present
on astrocytes and microglia [5-10], however the function of μ-opioid receptors on glial cells remains elusive.

A number of factors appear to contribute to the development of analgesic tolerance.
In general, the development of tolerance is thought to involve cellular adaptation/modulation
that results in decreased analgesic potency. The precise mechanism(s) of action is
not known; however, investigators have been able to attenuate or reverse established
analgesic tolerance to morphine by inhibiting either the release of neurotransmitters
and/or inhibition of their receptors [11-16]. Within the last decade, activation of spinal glia has emerged as a novel mechanism
underlying analgesic tolerance [17-19]. Relevant to the current study, the administration of sub-therapeutic (ultra-low)
doses of opioid specific antagonists (e.g. naloxone, naltrexone) augmented opioid-induced
analgesia and inhibited and/or reversed the development of tolerance and physical
dependence [20]. Although this relationship was studied intensively in various in vitro and in vivo models [20-22], only recently have clinical trials been undertaken to investigate the improved therapeutic
benefit of combining opioid analgesics with ultra-low dose opioid receptor antagonists.
To date, clinical trials have confirmed that combinations of opioids and ultra-low
dose antagonists both enhance and prolong opioid-induced analgesia, and prevent analgesic
tolerance and physical dependence [23,24]. Precisely how ultra-low dose antagonists prevent/reverse tolerance to opioid analgesics
is not fully understood, but spinal glia may play a crucial role. We demonstrate that
one contributing mechanism is that ultra-low dose naltrexone blocks opioid-induced
activation of spinal glial cells.

Results

Ultra-low dose naltrexone attenuated the development of tolerance to morphine antinociception

Chronic morphine does not induce cell proliferation

The number of GFAP and OX42-positive cells present in lumbar spinal cord sections
from animals chronically administered intrathecal vehicle (saline) or morphine (15
μg) were counted (Table 1). The number of GFAP-positive (astrocytes) and OX42-positive (microglia) cell bodies
observed in spinal cord sections from morphine-treated rats was not significantly
greater than the number in spinal cord sections from saline-treated controls. To confirm
this finding, cell proliferation was assessed via 5-bromo-deoxyuridine (100 mg/kg,
i.p; BrdU) experiments. BrdU was injected on alternative days 30 minutes prior to
intrathecal administration of saline or morphine (15 μg) for 5 days. Immunohistochemical
labelling of spinal cord sections collected from these animals revealed no significant
increase in the number of BrdU-positive cells in morphine-treated animals compared
to saline controls (Figure 5J). Double labelling of sections with the astrocytic marker GFAP (Figure 5A-C), or the neuronal marker MAP-2 (Figure 5G-I) revealed no co-localization with BrdU-positive cells. Iba1, the microglial and macrophage
marker, co-localized with a portion of the BrdU-positive cells (Figure 5D-F). The results of the BrdU experiments confirm the cell counts of astrocytes and microglia,
demonstrating that the chronic morphine treatment employed in this study does not
induce spinal cord cell proliferation.

Figure 5.The morphine-induced increase in astrocyte and microglial immuno-labelling is caused
by cell hypertrophy, not proliferation. Lumbar spinal cord sections were collected from animals administered BrdU (100 mg/kg)
by intraperitoneal injection on days 1, 3, 5, and either intrathecal vehicle (saline;
SAL) or morphine (15 μg; MS) once daily for five days by lumbar puncture. Representative
photomicrographs acquired by confocal microscopy of spinal cord sections double labelled
with 5-bromo-2-deoxyuridine (BrdU) and the astrocytic protein GFAP (B, C), the microglial
marker Iba1 (E, F) or the neuronal marker MAP2 (H, I). No co-localization of BrdU-positive
cells with GFAP or MAP-2-positive cells was observed. However, BrdU co-localized with
a small number of Iba1 positive cells (arrow), suggesting a small portion of the newly
formed cells were microglia or macrophages. (J). No difference was observed in the
number of BrdU-positive cells in the dorsal horn (lamina II-IV) of lumbar spinal cord
sections from animals administered chronic intrathecal saline or morphine. Data represent
means ± s.e.m. for n = 6 sections per rat from n = 3 per group. Statistical analyses
were performed by an un-paired t-test. ns = no significance. Scale bars, 30 μm.

Discussion

The current study has provided additional evidence that ultra-low dose naltrexone
attenuates the development of tolerance to the antinociceptive effects of morphine
as previously demonstrated by Powell et al [20]. As the mechanism by which this phenomenon occurs is unknown, this study sought to
investigate the contribution of glia in the actions of ultra-low dose opioid antagonists.
Intrathecal catheterization has been shown to induce gliosis [25], therefore the current study used lumbar puncture drug delivery to reproduce the
original behavioural findings of Powell et al [20]. Preliminary experiments investigated the effects of different ultra-low doses of
naltrexone on morphine tolerance. In this study, the dose of naltrexone (0.05 ng)
used in experiments by Powell et al [20] did not attenuate the loss in antinociception observed with chronic morphine administration.
However, a hundred-fold greater dose (5 ng) preserved the analgesic effects of morphine
throughout the treatment period, and thus was used to determine the effects on morphine-induced
gliosis. In addition to the use of intrathecal catheters for drug delivery, another
important difference in experimental protocol in the present study were the housing
conditions; animals used in this study were housed in a room on a reverse light-dark
cycle (lights off at 7:00 am), with all behavioural testing conducted during the animals'
active (dark) phase. It is well accepted that pain responsiveness and endogenous opioids
have circadian fluctuations in rats [26] and that morphine-induced antinociception is greater during the active phase compared
to during the inactive light phase. These fluctuations may account for the greater
dose of opioid antagonist required to attenuate tolerance in the present study compared
to what has been previously published.

Current research has demonstrated that spinal glia are not merely support cells within
the CNS as previously hypothesized (i.e. responsible for the maintenance of neurons
and CNS homeostasis); they also actively communicate with neurons, are involved in
the modulation of synaptic signalling and may be involved in the development of opioid
tolerance. Chronic, but not acute, morphine administration, induces gliosis characterized
by cell hypertrophy, and is associated with increased expression of GFAP [19,27-30] in astrocytes and CD3/CD11B (OX42) in microglia [31]. Reactive glial cells (microglia and astrocytes) can release a variety of pro-nociceptive
and neuroexcitatory substances (e.g. prostaglandins, excitatory amino acids, interleukins,
nitrogen oxide species, ATP, glutamate etc.), which may enhance pain transmission
by nociceptive neurons [19,28,32-34].

This is the first report to demonstrate that co-administration of ultra-low dose naltrexone
prevents morphine-induced gliosis, demonstrated by normalization of GFAP and CD3/CD11B
expression and attenuation of increased astrocyte cell volume. The observed increases
in GFAP/CD3/CD11B expression and astrocyte cell volume in spinal cord sections from
animals chronically administered intrathecal morphine are consistent with gliosis
and are in agreement with previous findings of astrocyte and microglial activation
by chronic morphine administration [19,28-30]. As no significant difference was found in the number of immuno-positive cells or
in the number of newly generated cells between morphine treated and saline controls,
glial proliferation likely contributes very little to the observed increases in GFAP
and CD3/CD11B expression. This finding is in agreement with that of Song and Zhao
[19], in which chronic morphine resulted in increased astrocyte immunoreactivity with
no difference in the number of cells from saline treated controls. In contrast, Narita
et al [30] reported that astrocyte proliferation was induced by chronic morphine administration;
however, no quantification of the number of GFAP-positive cells was reported. Agents
that modify [19] or inhibit [18] activation of astrocytes and microglia prevent the development of morphine tolerance;
thus inhibition of gliosis by ultra-low dose naltrexone may prevent the development
of analgesic tolerance. This evidence, taken in concert with the findings of the current
study, supports the hypothesis that spinal glia are involved in the development of
morphine analgesic tolerance and in the mediation of nociception. It has also been
reported that ultra-low dose naltrexone augments morphine antinociception in a model
of pertussis toxin induced hyperalgesia [35].

While the present study provides strong support for the role of glia in the ultra-low
dose effect, various molecular studies indicate that ultra-low dose antagonists may
prevent opioid receptor coupling to stimulatory G-proteins (Gs). Classically, opioid
activation of μ-opioid receptors results in coupling to inhibitory G-protein subunits
(Gi/Go) and produces analgesia; however, following chronic opioid administration,
increased coupling of μ-opioid receptors to Gs has been observed [21]. Therefore, increased excitatory stimulation via Gs-coupled μ-opioid receptors may
oppose the analgesic effects mediated via Gi/Go signalling, and manifest as tolerance
[21,36]. Wang et al [21] demonstrated that the switch in G-protein coupling to μ-opioid receptors induced
by chronic morphine could be prevented by co-administering an ultra-low dose of naloxone,
further supporting this hypothesis. Despite these advances, the switch in G-protein
coupling to μ-opioid receptors induced by chronic morphine treatment has not been
localized to a specific cell population within the spinal cord, and therefore, may
occur in glia or nociceptive neurons. On the contrary, the effects of ultra-low dose
antagonists may not be mediated by μ-opioid receptors but through a novel mechanism
such as an interaction with filamin A [37] or Toll-like receptors [38]. Thus, future studies will aim to identify the mechanism by which ultra-low dose
naltrexone alters gliosis.

Conclusions

The results of this study may have a significant impact on the clinical management
of moderate to severe pain. Patients currently treated with chronic opioid therapy
may benefit not only from increased efficacy of combined opioid treatment [23,39], but may also experience fewer and less severe adverse effects [24,40], as sufficient analgesia can be achieved and maintained at lower opioid doses. Additionally,
an understanding of the mechanism of action of opioid drugs will provide insight toward
the development of more selective and efficacious pharmacological treatments for pain
management. Not the least of which could be for improving treatment of chronic pain
conditions such as neuropathic pain where glial activation is also evident, with reactive
gliosis being a key contributor to the painful neuropathy [41-44]. Additionally, reduced opioid analgesic efficacy has also been reported in patients
with neuropathic pain [45,46], however, co-administration of ultra-low dose antagonists with opioid agonists increased
analgesic efficacy in animal models of neuropathic pain [47] and in clinical trials [23,24]. Future research will be required to determine if ultra-low dose naltrexone is able
to alleviate established chronic pain.

Methods

Animals

Adult male Sprague-Dawley rats (180-200 g; Charles River, Québec, Canada), were housed
in groups of two with ad libitum access to food and water, and maintained on a reverse 12/12 h light/dark cycle. All
behavioural experiments were performed during the dark phase of the cycle, and animals
were handled prior to experimentation in order to reduce stress-related analgesia.
All experimental protocols were approved by the Queen's University Animal Care Committee,
and complied with the policies and directives of the Canadian Council on Animal Care
and the International Association for the Study of Pain.

Behavioural tail flick assay

The effects of drug administration on thermal nociceptive responses were assessed
on Days 1, 3 and 5 of the study using the tail flick assay. In brief, a beam of radiant
light was applied to a spot marked 5 cm from the tip of the tail, and the latency
to a vigorous tail flick was measured. Three baseline latencies were measured prior
to drug injection to determine the normal nociceptive responses of the animals. A
cut-off time of three times the animal's average baseline was imposed to avoid tissue
damage in the event that the animal became unresponsive following drug injection.
Rats were then injected intrathecally with their respective treatments, and the thermal
latency measured at 30 minutes post-injection, as previous studies have found that
the peak antinociceptive effects of morphine occur at this time point [48]. Tail-flick values were converted to a maximum possible effect (% MPE): (post-drug
latency - baseline) ÷ (cut-off latency - baseline) × 100. Statistical analyses were
performed using a two-way analysis of variance (ANOVA), followed by Bonferroni's post-hoc multiple comparisons test to determine between group differences. P values less than
0.05 were considered significant. All behavioural testing was performed by the experimenter
blind to drug treatment.

Imaging of immunoreactive cells was performed as previously described [50]. In brief, immunoreactive cells were imaged using the Leica TCS SP2 multi photon
confocal microscope (Leica Microsystems Inc, Ontario, Canada). Images were taken within
the dorsal horn (lamina III-V) at 63× magnification for quantification of intensity.
Serial images (twenty-five to thirty-five) were captured at 100× magnification, at
0.75 μm increments throughout the z plane in the deep and superficial dorsal horn
(4 series per section, 3 sections per animal).

For quantification of the intensity of antibody labelling, images were converted to
gray scale using Adobe Photoshop 7.0. Using Image J (NIH), the mean gray values were
measured and the average within each treatment group calculated and expressed as mean
± s.e.m. For quantification of GFAP, OX42 and BrdU-positive cells, immunolabelled
cell bodies were counted for each section (150 μm × 150 μm) and the average within
each treatment group calculated and expressed as mean ± s.e.m. To quantify astrocyte
volume, images taken at 100× magnification were stacked and reconstructed in three-dimensions
using ImagePro Plus v5.0 software (MediaCybernetics, MD, USA). Total cell volume was
calculated for each reconstructed cell. The average volume for cells within each treatment
group was calculated and expressed as mean ± s.e.m. Mean intensities and 3D volumes
were analyzed by one-way ANOVA followed by Tukey's post-hoc multiple comparison test.
Differences in cell numbers were analyzed by unpaired T-tests. P values less than
0.05 were considered significant. All quantification data was collected by experimenter
blind to drug treatment.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

TAM participated in the design of the study, carried out data collection, statistical
analysis and interpretation, and drafted the manuscript. BM participated in the conception
and design of the study, data interpretation, and editing of the manuscript. CMC participated
in the conception and design of the study, data interpretation and editing of the
manuscript. All authors read and approved the final manuscript.

Acknowledgements

This work was supported by grants from the Canadian Institutes of Health Research
(CIHR) and the Canadian Foundation for Innovation awarded to CMC. CMC is a Canadian
Research Chair in chronic pain.

References

Cox BM: Molecular and cellular mechanisms in opioid tolerance. In Towards a New Pharmacotherapy of Pain. Edited by Bausbaum AI, Besson JM. John Wiley; 1999:137-156.